Dual role of GRHL3 in bladder carcinogenesis depending on histological subtypes.

The effect of grainyhead-like transcription factor 3 (GRHL3) on cancer development depends on the cancer subtypes as shown in tumor entities such as colorectal or oral squamous cell carcinomas. Here, we analyzed the subtype-specific role of GRHL3 in bladder carcinogenesis, comparing common urothelial carcinoma (UC) with squamous bladder cancer (sq-BLCA). We examined GRHL3 mRNA and protein expression in cohorts of patient samples, its prognostic role and its functional impact on tumorigeneses in different molecular and histopathological subtypes of bladder cancer. We showed for GRHL3 a reverse expression in squamous and urothelial bladder cancer subtypes. Stably GRHL3-overexpressing EJ28, J82, and SCaBER in vitro models revealed a tumor-suppressive function in squamous and an oncogenic role in the urothelial cancer cells affecting cell and colony growth, and migratory and invasive capacities. Transcriptomic profiling demonstrated highly subtype-specific GRHL3-regulated expression networks coined by the enrichment of genes involved in integrin-mediated pathways. In SCaBER, loss of ras homolog family member A (RHOA) GTPase activity was demonstrated to be associated with co-regulation of eukaryotic translation initiation factor 4E family member 3 (EIF4E3), a potential tumor suppressor gene. Thus, our data provide for the first time a detailed insight into the role of the transcription factor GRHL3 in different histopathological subtypes of bladder cancer.

Expressing Optogenetic Actuators Fused to N-terminal Mucin Motifs Delivers Targets to Specific Subcellular Compartments in Polarized Cells.

Optogenetics is a powerful approach in neuroscience research. However, other tissues of the body may benefit from controlled ion currents and neuroscience may benefit from more precise optogenetic expression. The present work constructs three subcellularly-targeted optogenetic actuators based on the channelrhodopsin ChR2-XXL, utilizing 5, 10, or 15 tandem repeats (TR) from mucin as N-terminal targeting motifs and evaluates expression in several polarized and non-polarized cell types. The modified channelrhodopsin maintains its electrophysiological properties, which can be used to produce continuous membrane depolarization, despite the expected size of the repeats. This work then shows that these actuators are subcellularly localized in polarized cells. In polarized epithelial cells, all three actuators localize to just the lateral membrane. The TR-tagged constructs also express subcellularly in cortical neurons, where TR5-ChR2XXL and TR10-ChR2XXL mainly target the somatodendrites. Moreover, the transfection efficiencies are shown to be dependent on cell type and tandem repeat length. Overall, this work verifies that the targeting motifs from epithelial cells can be used to localize optogenetic actuators in both epithelia and neurons, opening epithelia processes to optogenetic manipulation and providing new possibilities to target optogenetic tools.

Single-neuron mechanical perturbation evokes calcium plateaus that excite and modulate the network.

Mechanical stimulation is a promising means to non-invasively excite and modulate neuronal networks with a high spatial resolution. Despite the thorough characterization of the initiation mechanism, whether or how mechanical responses disperse into non-target areas remains to be discovered. Our in vitro study demonstrates that a single-neuron deformation evokes responses that propagate to about a third of the untouched neighbors. The responses develop via calcium influx through mechanosensitive channels and regeneratively propagate through the neuronal ensemble via gap junctions. Although independent of action potentials and synapses, mechanical responses reliably evoke membrane depolarizations capable of inducing action potentials both in the target and neighbors. Finally, we show that mechanical stimulation transiently potentiates the responding assembly for further inputs, as both gain and excitability are transiently increased exclusively in neurons that respond to a neighbor's mechanical stimulation. The findings indicate a biological component affecting the spatial resolution of mechanostimulation and point to a cross-talk in broad-network mechanical stimulations. Since giga-seal formation in patch-clamp produces a similar mechanical stimulus on the neuron, our findings inform which neuroscientific questions could be reliably tackled with patch-clamp and what recovery post-gigaseal formation is necessary.

Elastomeric Pillar Cages Modulate Actomyosin Contractility of Epithelial Microtissues by Substrate Stiffness and Topography.

Cell contractility regulates epithelial tissue geometry development and homeostasis. The underlying mechanobiological regulation circuits are poorly understood and experimentally challenging. We developed an elastomeric pillar cage (EPC) array to quantify cell contractility as a mechanoresponse of epithelial microtissues to substrate stiffness and topography. The spatially confined EPC geometry consisted of 24 circularly arranged slender pillars (1.2 MPa, height: 50 µm; diameter: 10 µm, distance: 5 µm). These high-aspect-ratio pillars were confined at both ends by planar substrates with different stiffness (0.15-1.2 MPa). Analytical modeling and finite elements simulation retrieved cell forces from pillar displacements. For evaluation, highly contractile myofibroblasts and cardiomyocytes were assessed to demonstrate that the EPC device can resolve static and dynamic cellular force modes. Human breast (MCF10A) and skin (HaCaT) cells grew as adherence junction-stabilized 3D microtissues within the EPC geometry. Planar substrate areas triggered the spread of monolayered clusters with substrate stiffness-dependent actin stress fiber (SF)-formation and substantial single-cell actomyosin contractility (150-200 nN). Within the same continuous microtissues, the pillar-ring topography induced the growth of bilayered cell tubes. The low effective pillar stiffness overwrote cellular sensing of the high substrate stiffness and induced SF-lacking roundish cell shapes with extremely low cortical actin tension (11-15 nN). This work introduced a versatile biophysical tool to explore mechanobiological regulation circuits driving low- and high-tensional states during microtissue development and homeostasis. EPC arrays facilitate simultaneously analyzing the impact of planar substrate stiffness and topography on microtissue contractility, hence microtissue geometry and function.

From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion.

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM-ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM-ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.

Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini.

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs' barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.

The ECM Modulator ITIH5 Affects Cell Adhesion, Motility and Chemotherapeutic Response of Basal/Squamous-Like (BASQ) Bladder Cancer Cells.

This study aims at characterizing the role of the putative tumor suppressor ITIH5 in basal-type bladder cancers (BLCA). By sub-classifying TCGA BLCA data, we revealed predominant loss of ITIH5 expression in the basal/squamous-like (BASQ) subtype. ITIH5 expression inversely correlated with basal-type makers such as KRT6A and CD44. Interestingly, Kaplan-Meier analyses showed longer recurrence-free survival in combination with strong CD44 expression, which is thought to mediate ITIH-hyaluronan (HA) binding functions. In vitro, stable ITIH5 overexpression in two basal-type BLCA cell lines showing differential CD44 expression levels, i.e., with (SCaBER) and without squamous features (HT1376), demonstrated clear inhibition of cell and colony growth of BASQ-type SCaBER cells. ITIH5 further enhanced HA-associated cell-matrix attachment, indicated by altered size and number of focal adhesion sites resulting in reduced cell migration capacities. Transcriptomic analyses revealed enrichment of pathways and processes involved in ECM organization, differentiation and cell signaling. Finally, we provide evidence that ITIH5 increase sensitivity of SCaBER cells to chemotherapeutical agents (cisplatin and gemcitabine), whereas responsiveness of HT1376 cells was not affected by ITIH5 expression. Thus, we gain further insights into the putative role of ITIH5 as tumor suppressor highlighting an impact on drug response potentially via the HA-CD44 axis in BASQ-type BLCA.

The Basement Membrane in a 3D Breast Acini Model Modulates Delivery and Anti-Proliferative Effects of Liposomal Anthracyclines.

Breast cancer progression is marked by cancer cell invasion and infiltration, which can be closely linked to sites of tumor-connected basement membrane thinning, lesion, or infiltration. Bad treatment prognosis frequently accompanies lack of markers for targeted therapy, which brings traditional chemotherapy into play, despite its adverse effects like therapy-related toxicities. In the present work, we compared different liposomal formulations for the delivery of two anthracyclines, doxorubicin and aclacinomycin A, to a 2D cell culture and a 3D breast acini model. One formulation was the classical phospholipid liposome with a polyethylene glycol (PEG) layer serving as a stealth coating. The other formulation was fusogenic liposomes, a biocompatible, cationic, three-component system of liposomes able to fuse with the plasma membrane of target cells. For the lysosome entrapment-sensitive doxorubicin, membrane fusion enabled an increased anti-proliferative effect in 2D cell culture by circumventing the endocytic route. In the 3D breast acini model, this process was found to be limited to cells beneath a thinned or compromised basement membrane. In acini with compromised basement membrane, the encapsulation of doxorubicin in fusogenic liposomes increased the anti-proliferative effect of the drug in comparison to a formulation in PEGylated liposomes, while this effect was negligible in the presence of intact basement membranes.

Nanoscale Topography and Poroelastic Properties of Model Tissue Breast Gland Basement Membranes.

Basement membranes (BMs) are thin layers of condensed extracellular matrix proteins serving as permeability filters, cellular anchoring sites, and barriers against cancer cell invasion. It is believed that their biomechanical properties play a crucial role in determining cellular behavior and response, especially in mechanically active tissues like breast glands. Despite this, so far, relatively little attention has been dedicated to their analysis because of the difficulty of isolating and handling such thin layers of material. Here, we isolated BMs derived from MCF10A spheroids-three-dimensional breast gland model systems mimicking in vitro the most relevant phenotypic characteristics of human breast lobules-and characterized them by atomic force microscopy, enhanced resolution confocal microscopy, and scanning electron microscopy. By performing atomic force microscopy height-clamp experiments, we obtained force-relaxation curves that offered the first biomechanical data on isolated breast gland BMs to our knowledge. Based on enhanced resolution confocal microscopy and scanning electron microscopy imaging data, we modeled the system as a polymer network immersed in liquid and described it as a poroelastic material. Finite-element simulations matching the experimental force-relaxation curves allowed for the first quantification, to our knowledge, of the bulk and shear moduli of the membrane as well as its water permeability. These results represent a first step toward a deeper understanding of the mechanism of tensional homeostasis regulating mammary gland activity as well as its disruption during processes of membrane breaching and metastatic invasion.

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients.

Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.

ITIH5 mediates epigenetic reprogramming of breast cancer cells.

BACKGROUND: Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive. METHODS: ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement. RESULTS: ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards ß1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility. CONCLUSIONS: Our results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis.

BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. METHODS: Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. RESULTS: SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer. CONCLUSIONS: The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation.

Dual role of macrophage migration inhibitory factor (MIF) in human breast cancer.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine and mediator of acute and chronic inflammatory diseases. MIF is overexpressed in various tumours and has been suggested as a molecular link between chronic inflammation and cancer. MIF overexpression is observed in breast cancer but its causal role in the development of this tumour entity is unclear. METHODS: MIF levels in breast cancer cell lines were determined by ELISA and Western blot. CD74 was measured by Western blot, fluorescence microscopy and flow cytometry. Cell proliferation was studied by BrdU incorporation, cell adhesion by Matrigel adhesion assay, and cell invasion by migration assay through Matrigel-coated filters using the Transwell system. MIF expression in primary human breast cancers was measured by tissue microarray and a semi-quantitative immunoreactivity score (IRS) and comparison with histopathological parameters and patient outcome data. RESULTS: MIF was abundantly expressed in the non-invasive breast cancer cell lines MDA-MB-468 and ZR-75-1, but not in invasive MDA-MB-231 cells, which in turn expressed higher levels of the MIF-receptor CD74. Stimulation with exogenous MIF led to a dramatic upregulation of MIF secretion (50-fold) in MDA-MB-231 cells. Autocrine MIF promoted tumour cell proliferation, as indicated by blockade of MIF or CD74 in MDA-MB-231 and MDA-MB-468, and MDA-MB-231 invasiveness was enhanced by exogenous MIF. We correlated the expression of MIF with histopathological parameters and patient outcome data, using a tissue microarray of 175 primary invasive breast cancers and 35 normal control tissues. MIF was upregulated in breast cancer versus normal tissue (median IRS = 8 versus 6). MIF expression showed positive correlations with progesterone (p = 0.006) and estrogen (p = 0.028) receptor expression, markers of a favourable prognosis and a negative correlation to tumour size (p = 0.007). In line with these data, disease-specific overall (OS) as well as recurrence-free (RFS) survival was significantly improved in breast cancer patients with abundant cytosolic MIF expression compared to MIF low expressers (5-year OS = 67% versus 50%, p = 0.0019; 5-year RFS = 52% versus 36%, p = 0.0327). CONCLUSION: We conclude that intracellular expression of MIF in breast cancer cells is beneficial, whereas extracellular MIF may play a pro-oncogenic role in promoting breast cancer cell-stroma interactions.

Promoter hypermethylation of the SFRP2 gene is a high-frequent alteration and tumor-specific epigenetic marker in human breast cancer.

BACKGROUND: We have previously reported that expression of the Wnt antagonist genes SFRP1 and SFRP5 is frequently silenced by promoter hypermethylation in breast cancer. SFRP2 is a further Wnt inhibitor whose expression was recently found being downregulated in various malignancies. Here we investigated whether SFRP2 is also implicated in human breast cancer, and if so whether SFRP2 promoter methylation might serve as a potential tumor biomarker. METHODS: We analyzed SFRP2 mRNA expression and SFRP2 promoter methylation in 10 breast cell lines, 199 primary breast carcinomas, 20 matched normal breast tissues and 17 cancer-unrelated normal breast tissues using RT-PCR, realtime PCR, methylation-specific PCR and Pyrosequencing, respectively. SFRP2 protein expression was assessed by immunohistochemistry on a tissue microarray. Proliferation assays after transfection with an SFRP2 expression vector were performed with mammary MCF10A cells. Statistical evaluations were accomplished with SPSS 14.0 software. RESULTS: Of the cancerous breast cell lines, 7/8 (88%) lacked SFRP2 mRNA expression due to SFRP2 promoter methylation (P < 0.001). SFRP2 expression was substantially restored in most breast cell lines after treatment with 5-aza-2'-deoxycytidine and trichostatin A. In primary breast carcinomas SFRP2 protein expression was strongly reduced in 93 of 125 specimens (74%). SFRP2 promoter methylation was detected in 165/199 primary carcinomas (83%) whereas all cancer-related and unrelated normal breast tissues were not affected by SFRP2 methylation. SFRP2 methylation was not associated with clinicopathological factors or clinical patient outcome. However, loss of SFRP2 protein expression showed a weak association with unfavorable patient overall survival (P = 0.071). Forced expression of SFRP2 in mammary MCF10A cells substantially inhibited proliferation rates (P = 0.045). CONCLUSION: The SFRP2 gene is a high-frequent target of epigenetic inactivation in human breast cancer. Its methylation leads to abrogation of SFRP2 expression, conferring a growth advantage to epithelial mammary cells. This altogether supports a tumor suppressive function of SFRP2. Although clinical patient outcome was not associated with SFRP2 methylation, the high frequency of this epimutation and its putative specificity to neoplastic cells may qualify SFRP2 promoter methylation as a potential candidate screening marker helping to improve early breast cancer detection.

The ubiquitin-like molecule interferon-stimulated gene 15 (ISG15) is a potential prognostic marker in human breast cancer.

INTRODUCTION: ISG15 is an ubiquitin-like molecule that is strongly upregulated by type I interferons as a primary response to diverse microbial and cellular stress stimuli. However, alterations in the ISG15 signalling pathway have also been found in several human tumour entities. To the best of our knowledge, in the current study we present for the first time a systematic characterisation of ISG15 expression in human breast cancer and normal breast tissue both at the mRNA and protein level. METHOD: Using semiquantitative real-time PCR, cDNA dot-blot hybridisation and immunohistochemistry, we systematically analysed ISG15 expression in invasive breast carcinomas (n = 910) and normal breast tissues (n = 135). ISG15 protein expression was analysed in two independent cohorts on tissue microarrays; in an initial evaluation set of 179 breast carcinomas and 51 normal breast tissues; and in a second large validation set of 646 breast carcinomas and 10 normal breast tissues. In addition, a collection of benign and malignant mammary cell lines (n = 9) were investigated for ISG15 expression. RESULTS: ISG15 was overexpressed in breast carcinoma cells compared with normal breast tissue, both at the RNA and protein level. Recurrence-free (p = 0.030), event-free (p = 0.001) and overall (p = 0.001) survival analyses showed a significant correlation between ISG15 overexpression and unfavourable prognosis. CONCLUSION: Therefore, ISG15 may represent a novel breast tumour marker with prognostic significance and may be helpful in selecting patients for and predicting response to the treatment of human breast cancer.

Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer.

BACKGROUND: Inhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. METHODS: ID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters. RESULTS: Frequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level. Interestingly, ID4 promoter methylation was a factor for unfavourable recurrence-free survival (P=0.036) and increased risk for lymph node metastasis (P=0.030). CONCLUSION: ID4 is indeed a novel tumour suppressor gene in normal human breast tissue and is epigenetically silenced during cancer development, indicating increased risk for tumour relapse. Thus, ID4 methylation status could serve as a prognostic biomarker in human breast cancer.

Epigenetic inactivation of the secreted frizzled-related protein-5 (SFRP5) gene in human breast cancer is associated with unfavorable prognosis.

Disruption of the Wnt pathway is thought to be crucial in the development of human cancer. Pathway inhibitory members of the secreted frizzled-related protein (SFRP) family were found to be downregulated due to epigenetic inactivation in various malignancies. To date, only SFRP1 has been studied in human breast cancer and we questioned whether other SFRP genes may be implicated in the pathogenesis of this disease as well. An initial real-time polymerase chain reaction analysis of SFRP5 expression in normal human tissues (n = 9) revealed weak expression in most tissues, including breast. Malignant mammary cell lines showed further SFRP5 expression loss in five of six cases. Consistently, in matched pairs of primary breast tumor/normal breast tissue, this downregulation (>5-fold) could be confirmed (n = 8/13; 62%). We identified promoter methylation as the predominant mechanism of SFRP5 gene silencing since SFRP5 promoter methylation correlated significantly with loss of SFRP5 expression in cell lines (P = 0.040) and primary tumors (P = 0.003). Moreover, cancerous cell lines re-expressed SFRP5 messenger RNA following treatment with DNA-demethylating drugs. Of 168 primary breast carcinomas, 73% harbored a methylated SFRP5 promoter, whereas 27% were unaffected by epigenetic alteration. Most interestingly, SFRP5 methylation was associated with reduced overall survival (OS) (P = 0.045) and was an independent risk factor affecting OS in a multivariate Cox proportional hazard model (hazard ratio): 4.55; 95% confidence interval: 1.01-20.56; P = 0.049). In conclusion, SFRP5 is a target of epigenetic inactivation in human breast cancer, supporting the hypothesis of its role as tumor suppressor gene. SFRP5 methylation may be a novel DNA-based biomarker potentially useful in clinical breast cancer management.